The putative natural killer decoy early gene m04 (gp34) of murine cytomegalovirus encodes an antigenic peptide recognized by protective antiviral CD8 T cells

Abstract

Several early genes of murine cytomegalovirus (MCMV) encode proteins that mediate immune evasion by interference with the major histocompatibility complex class I (MHC-I) pathway of antigen presentation to cytolytic T lymphocytes (CTL). Specifically, the m152 gene product gp37/40 causes retention of MHC-I molecules in the endoplasmic reticulum (ER)-Golgi intermediate compartment. Lack of MHC-I on the cell surface should activate natural killer (NK) cells recognizing the "missing self." The retention, however, is counteracted by the m04 early gene product gp34, which binds to folded MHC-I molecules in the ER and directs the complex to the cell surface. It was thus speculated that gp34 might serve to silence NK cells and thereby complete the immune evasion of MCMV. In light of these current views, we provide here results demonstrating an in vivo role for gp34 in protective antiviral immunity. We have identified an antigenic nonapeptide derived from gp34 and presented by the MHC-I molecule D(d). Besides the immunodominant immediate-early nonapeptide consisting of IE1 amino acids 168-176 (IE1(168-176)), the early nonapeptide m04(243-251) is the second antigenic peptide described for MCMV. The primary immune response to MCMV generates significant m04-specific CD8 T-cell memory. Upon adoptive transfer into immunodeficient recipients, an m04-specific CTL line controls MCMV infection with an efficacy comparable to that of an IE1-specific CTL line. Thus, gp34 is the first noted early protein of MCMV that escapes viral immune evasion mechanisms. These data document that MCMV is held in check by a redundance of protective CD8 T cells recognizing antigenic peptides in different phases of viral gene expression.

FIG. 1

State-of-the-art models of IE1 presentation and evasion. (Left) IE1 presentation model, based on references , , , and . In the IE phase, and in particular under IE conditions enforced by the metabolic inhibitors cycloheximide and actinomycin D, the IE1 protein pp89 encoded by gene ie1 is expressed and processed, and the nonapeptide YPHFMPTNL is presented in the haplotype H-2^d by the MHC-I molecule L^d. (Right) IE1 immune evasion model, based on references , , , , and . In the E phase of viral gene expression, gp37/40 encoded by gene m152 causes the retention of peptide-loaded MHC-I molecules in the ER-Golgi intermediate compartment (ERGIC) and cis-Golgi, thereby preventing the presentation of the IE1 peptide. It is proposed that lack of MHC-I surface expression renders the E-phase cells susceptible to recognition by NK cells.

FIG. 2

Antigenic activities in HPLC fractions derived from productively infected MEF. (A) Elution profile of the HPLC. The dotted line describes the acetonitrile gradient from 0 to 80%. OD₂₂₀, optical density at 220 nm. (B) Detection of naturally processed antigenic peptides in HPLC fractions by short-term microculture CTLL generated from MCMV-specific memory spleen cells by repeated restimulation with the corresponding HPLC fractions. The cytolytic assay was performed with P815 target cells. Data represent the mean of triplicates. Fractions 27 and 28 contain the already known IE1 peptide. The arrow points to fraction 22, which represents the most consistently observed novel antigenic activity. The dashed line represents the baseline of the assay, as defined with no HPLC fraction added to the cultures. (C) MHC-I restriction analysis for the antigenic activities in HPLC fractions, performed with MHC-I transfectants L-K^d, L-D^d, and L-L^d as target cells. Data represent the mean of triplicates. The arrow marks the D^d-restricted activity in fraction 22.

FIG. 3

Identification of an antigenic peptide by screening of a D^d motif peptide library of MCMV. (A) A search for the D^d binding motif xGPxxxxx[L, I, F] (44) was performed for all open reading frames of the full-length genomic sequence of MCMV (45), and corresponding synthetic nonapeptides were used at the indicated concentrations for the generation of short-term microculture CTLL by repeated restimulation of MCMV-specific memory spleen cells. The cytolytic assay was performed with P815 target cells. Data represent the cytolytic activity in individual microcultures. The genomic positions of the nonapeptide-coding sequences are given by the positions of the first nucleotides according to the listing by Rawlinson et al. (45). C, complementary strand. (B) Map of the location of the prominent antigenic peptide (nucleotide [n] positions 3996 to 4022), representing aa 243 to 251 of gp34 encoded by gene m04 (31). TM, transmembrane region; CY, cytoplasmic tail. The peptide sequence is given in one-letter code.

FIG. 4

Screening of an MHC-I motif peptide library of gene m04 by ELISPOT assays. (A) Searches for the K^d motif x[Y, F]xxxxxx[I, L, V] and the L^d motif x[P, S]xxxxxx[F, L, M] (44) were performed for the m04 open reading frame. Besides the single D^d motif (Fig. 3), a single K^d motif and 11 L^d motifs were found. The locations of the motifs within the gp34 sequence are indicated by bars. The asterisk marks the single K^d motif. For abbreviations, see the legend to Fig. 3. (B) Corresponding synthetic peptides were tested in an IFN-γ-based ELISPOT assay performed with spleen cells derived from acutely infected BALB/c mice at 2 weeks after infection (open bars) or from memory spleen cells (pool 1) derived from three latently infected BALB/c mice at 4 months after infection (filled bars). (C) IFN-γ-based ELISPOT assay performed with spleen cells from adult, unprimed BALB/c mice (open bars) or from memory spleen cells (independent pool 2) derived from another three latently infected BALB/c mice at 4 months after infection (filled bars). The arrow points to results obtained for peptide 243-251. The asterisk marks the peptide that corresponds to the K^d motif. Controls from left to right: (i) spleen cells (SC) and P815-B7 as PPC, but no peptide added; (ii) SC only; (iii) SC, PPC, and, as a positive reference, the IE1 nonapeptide YPHFMPTNL. Throughout, peptides were used at a concentration of 10⁻⁸ M.

FIG. 5

Kinetics of MCMV gene expression in MEF. Poly(A)⁺ RNA, derived from infected MEF harvested at the indicated time points after infection, was subjected to RT-PCRs specific for the indicated genes, including cellular hprt as a control. First lane, all reagents except RT; last lane, poly(A)⁺ RNA derived from MEF infected for 24 h in the presence of PAA (250 μg per ml). Shown are autoradiographs obtained after agarose gel electrophoresis, Southern blotting, and hybridization with the respective γ-³²P-end-labeled oligonucleotide probes.

FIG. 6

Generation of m04-CTLL. Memory spleen cells derived from BALB/c mice at 4 months after infection with MCMV were restimulated under bulk culture conditions with synthetic m04 peptide YGPSLYRRF at the molar concentrations indicated. Panels A to C show cytolytic activity of CTLL after one to three rounds of restimulation, respectively. The cytolytic assay was performed at an E/T ratio of 15:1. Target cells were P815 mastocytoma cells pulsed with the m04 peptide at the molar concentrations indicated.

FIG. 7

Properties of m04-CTLL and IE1-CTLL in vitro. (A) Comparison of cytolytic effector function. (Left) Peptide dose dependence of target cell recognition. Target cells were P815 mastocytoma cells pulsed with the indicated molar concentrations of the appropriate synthetic peptide. The assays were performed at an E/T cell ratio of 15:1. The 100-fold peptide molarity difference between the two CTLL is highlighted by a two-headed arrow. (Right) Cytolytic potential of the two CTLL, compared by E/T titration. P815 mastocytoma target cells were pulsed with optimal molar concentrations of synthetic peptide, 10⁻⁷ and 10⁻⁸ M for m04-CTLL and IE1-CTLL, respectively. (B) Detection of intracellular IFN-γ by two-color cytofluorometric analysis. Production of IFN-γ was stimulated in IE1-CTLL and m04-CTLL by sensitization with 10⁻⁶ M synthetic peptide, with the heterologous peptides serving as negative controls. CTL were stained for the expression of IFN-γ (fluorescein fluorescence [FL-1]; abscissa) and CD8 (PE fluorescence, FL-2; ordinate). Quadrants (dotted lines) were defined by omission of PE-conjugated specific antibody in the case of CD8 and by a fluorescein-conjugated isotype control antibody in the case of IFN-γ. All cells of both CTLL expressed CD8. Data obtained with 25,000 and 12,500 cells for IE1-CTLL and m04-CTLL, respectively, are shown as contour plots in a 50% log mode.

FIG. 8

Control of hepatic MCMV infection by liver-infiltrating CTL. (A) Two-color immunohistological analysis visualizing infected hepatocytes (red intranuclear staining) and liver-infiltrating CD3ɛ-expressing T cells (black membrane staining). The indicated cell numbers of m04-specific CTL (from the same CTLL as characterized in Fig. 7) were adoptively transferred into immunocompromised, infected BALB/c recipients. ∅, control recipients with no cell transfer. The analysis was performed on day 12 after infection and cell transfer. (a to d) Overviews. The arrows point to sites of interest shown enlarged in corresponding panels (a* to d*). Bar markers represent 50 μm. (B) Quantitative two-color immunohistology after adoptive transfer of m04-CTLL (left, corresponding to panel A) or of IE1-CTLL (right). The numbers of infected hepatocytes (red dots) and of infiltrating T lymphocytes (black dots) refer to representative 10-mm² areas of liver tissue sections. Each dot represents an individual transfer recipient. Median values are marked by a horizontal bar.

FIG. 9

Control of MCMV replication in further organs relevant to CMV disease. Infectious virus in the indicated organs of immunocompromised BALB/c recipients (same experiment as in Fig. 8) was quantitated by an in vitro plaque assay performed on day 12 after adoptive transfer of the indicated cell numbers of m04-CTL (left) or of IE1-CTL (right). ∅, control recipients with no cell transfer. Virus titers are given as PFU* determined under conditions of centrifugal enhancement of infectivity. Each dot represents an individual transfer recipient. Median values are marked by a horizontal bar. DL and dotted line, detection limit of the plaque assay.

FIG. 10

State-of-the-art models of the role of gp34 in the E phase. (Left) NK cell decoy model, based on reference . The m04 E-gene product gp34 binds to MHC-I molecules in the ER and directs the complex to the cell surface. It is proposed that restoration of MHC-I surface expression silences the NK cells. (Right) Model of gp34 antigen presentation in the E phase, based on this report. The E-phase protein gp34 is by itself subject of antigen processing, and peptide YGPSLYRRF is presented in the haplotype H-2^d by the MHC-I molecule D^d. Although not documented for cultured fibroblasts, the priming of memory T cells and the antiviral function of m04-CTL imply peptide presentation in infected tissue cells in vivo. It remains open to question whether the gp34 peptide-presenting MHC-I molecules are also complexed with native gp34 protein.